Mesenchymal stem cells for the treatment of cns diseases

ABSTRACT

An isolated human cell is disclosed comprising at least one mesenchymal stem cell phenotype and secreting brain-derived neurotrophic factor (BDNF), wherein a basal secretion of the BDNF is at least five times greater than a basal secretion of the BDNF in a mesenchymal stem cell. Methods of generating same and uses of same are also disclosed.

RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.15/261,959 filed on Sep. 11, 2016 which is a division of U.S. patentapplication Ser. No. 14/556,281 filed on Dec. 1, 2014, now U.S. Pat. No.9,474,787 which is a division of U.S. patent application Ser. No.14/164,286 filed on Jan. 27, 2014, now U.S. Pat. No. 8,900,574 which isa division of U.S. patent application Ser. No. 12/994,761 filed on Nov.25, 2010, now U.S. Pat. No. 8,663,987 which is a National Phase of PCTPatent Application No. PCT/IL2009/000525 having International FilingDate of May 26, 2009, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/071,970 filed on May 28, 2008. Thecontents of the above Applications are all incorporated herein byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to cells and populations thereof which canbe used for treating CNS diseases.

Parkinson's disease is an age-related disorder characterized byprogressive loss of dopamine producing neurons in the substantia nigraof the midbrain, which in turn leads to progressive loss of motorfunctions manifested through symptoms such as tremor, rigidity andataxia.

The use of stem cells as a cellular source in cell replacement therapyfor Parkinson's disease has been suggested. Stem cells have the abilityto exist in vivo in an undifferentiated state and to self-renew. Theyare not restricted to cell types specific to the tissue of origin, andso they are able to differentiate in response to local environmentalcues from other tissues. This capability of self renewal anddifferentiation has great therapeutic potential in curing diseases.

U.S. Patent Appl. 20050265983 to the present inventors teaches humandopamine synthesizing MSCs which express neuronal markers andtranscription factors that characterize midbrain DA neuron followinginduction of neuronal differentiation.

As an alternative to a dopamine replacement strategy, cell therapy maybe aimed at restoring or reestablishing the normal anatomy(connectivity) and physiology (appropriate synaptic contacts andfunctioning) of the striatum. In this instance, the grafted cells haveto survive and possess morphological electrophysiological and functionaldopaminergic properties.

Neurotrophic factors (NTFs) are secreted proteins that regulate thesurvival, functional maintenance and phenotypic development of neuronalcells. Alterations in NTF levels are involved in triggering programmedcell-death in neurons and thus contribute to the pathogenesis ofParkinson's and other neurodegenerative diseases.

One of the most potent NTF for dopaminergic neurons is called glial cellline-derived neurotrophic factor (GDNF). It is known to promote thesurvival of the dopaminergic neurons in the substantia nigra, promoteneurite outgrowth, increase cell body size and also raise levels of TH.GDNF belongs to a family of proteins, related to the TGF-β-superfamily,currently consisting of four neurotrophic factors: GDNF, Neurturin(NTN), Persephin, and Artemin/Neublastin. These factors are known toserve as regulators of cell proliferation and differentiation.

Various cells type produce GDNF including glia cells (oligodendrocytesand astrocyte), neuroblastoma and glioblastoma cell lines. It hasrecently been shown that rat BMSCs cultured in DMEM supplemented with20% fetal bovine serum, at passage 6 express GDNF and NGF [Garcia R, etal., Biochem Biophys Res Commun. 316(3):753-4, 2004].

Administration of GDNF directly into the brain has been shown to beeffective in various animal models of PD. In addition, exposure of cellsto GDNF prior to transplant has proven beneficial. For instance,grafting of 400,000 fetal dopaminergic neurons prior to transplantationsignificantly improved the rotational behavior of lesioned rats [MehtaV, et al., J Neurosurg. 1999 Apr; 90(4):804-6].

Various methods have been used to aid administration of GDNF into thebrain including osmotic pumps, capsules and microspheres. Anotherapproach for GDNF delivery is in vivo gene therapy. Bone marrowmesenchymal cells genetically engineered to express GDNF, transplantedinto MPTP-lesioned mice, were able to protect nigral neurons as well asstriatal fibers [Park, K., Neurosci. Res. 40: 315-323, 2001].

Glutamate is the main excitatory amino acid neurotransmitter in thehuman central nervous system (CNS). It plays a major role in synapticplasticity, learning, development, cognitive functions and humanbehavior. However, if not properly controlled glutamate may lead todetrimental results. Prolonged exposure to glutamate leads to overstimulation of excitatory a.a receptors, a process culminating inneuronal cell death.

Regulation of glutamate levels near and within the synaptic cleft isprimarily performed by astrocytes. When extracellular glutamate levelsare high, astrocytes can remove it from the synaptic space. Glutamateuptake is facilitated mainly by high affinity excitatory a.atransporters, which insert Na⁺ and H⁺ into the cell, while removing K⁺from the cell, thus enabling the transfer of glutamate into the cellagainst its electrochemical gradient. Although, the less common form ofNa⁺ independent transport also occurs.

Accumulating evidence implicate glutamate toxicity in thepathophysiology of several acute neurodegenerative processes, mainlycerebral ischemia and traumatic brain injuries. Furthermore, it appearsthat glutamate toxicity participates in chronic neurodegenerativedisorders such as Huntington's disease (HD), Parkinson's disease (PD),amyotrophic lateral sclerosis (ALS), epilepsy and Alzheimer's disease(AD).

Reducing extracellular glutamate levels around the susceptible neuronsaffected by glutamate toxicity in the different disease modules may haltthe neurodegenerative progression. A possible approach to provide suchneuronal protection is by transplanting cells capable of performingglutamate uptake adjacent to the endangered neurons. Adult humanmesenchymal stem cells (hMSC) obtained from bone marrow, may vary wellprove to be a viable source for such transplantations.

Several studies have shown that MSCs following exposure to differentfactors in vitro, change their phenotype and demonstrate neuronal andglial markers [Kopen, G. C., et al., Proc Natl Acad USA. 96(19):10711-6,1999; Sanchez-Ramos, et al. Exp Neurol. 164(2):247-56. 2000; Woodbury,D., J Neurosci Res. 61(4):364-70,2000; Woodbury, D., et al., J NeurosciRes. 69(6):908-17, 2002; Black, I. B., Woodbury, D. Blood Cells Mol Dis.27(3):632-6, 2001; Kohyama, J., et al. Differentiation. 68(4-5):235-44,2001; Levy, Y. S. J Mol Neurosci. 21(2):121-32, 2003].

WO2006/134602 teaches differentiation protocols for the generation ofneurotrophic factor secreting cells.

WO2007/066338 teaches differentiation protocols for the generation ofoligodendrocyte-like cells.

WO2004/046348 teaches differentiation protocols for the generation ofneuronal-like cells.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated human cell comprising at least onemesenchymal stem cell phenotype and secreting brain-derived neurotrophicfactor (BDNF), wherein a basal secretion of the BDNF is at least fivetimes greater than a basal secretion of the BDNF in a mesenchymal stemcell.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell population comprising humanmesenchymal stem cells, wherein at least 50% of the cells express glialfibrillary acidic protein (GFAP) and secrete at least one neurotrophicfactor.

According to some embodiments of the invention, the at least oneneurotrophic factor is glial cell line-derived neurotrophic factor(GDNF) or BDNF.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell population comprising human cellswherein:

-   -   (i) at least N % of the human cells secreting brain-derived        neurotrophic factor (BDNF), wherein a basal secretion of the        BDNF is at least five times greater than a basal secretion of        the BDNF in a mesenchymal stem cell;    -   (ii) at least M % of the human cells comprise at least one        mesenchymal stem cell phenotype; and    -   (iii) at least one of the human cells secretes the BDNF and the        at least one mesenchymal stem cell phenotype;    -   where M and N are each independently selected between 1 and 99.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeagent the cell populations of the present invention, and apharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating cells useful for treating a CNSdisease or disorder, the method comprising:

-   -   (a) incubating mesenchymal stem cells in a culture medium        comprising platelet lysate to generate propagated mesenchymal        stem cells; and    -   (b) incubating the propagated mesenchymal stem cells in a        differentiating medium, thereby generating cells useful for        treating the CNS disease or disorder.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell generated according to the method ofthe present invention, having an astrocyte phenotype.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell generated according to the method ofthe present invention, having an oligodendrocyte phenotype.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell generated according to the method ofthe present invention, secreting dopamine.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating cells secreting neurotrophicfactors, comprising

-   -   (a) incubating mesenchymal stem cells in a serum free medium        comprising platelet lysate to generate propagated mesenchymal        stem cells; and    -   (b) incubating the propagated mesenchymal stem cells in a        differentiating medium comprising at least one differentiating        agent, the at least one differentiating agent being selected        from the group consisting of platelet derived growth factor        (PDGF), human neuregulin 1-β1, FGF2, EGF, N2, IBMX and cAMP,        thereby generating cells secreting neurotrophic factors.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a CNS disease or disordercomprising administering to an individual in need thereof atherapeutically effective amount of a cell population generatedaccording to the method of claim 18, thereby treating the CNS disease ordisorder.

According to some embodiments of the invention, the isolated human celltakes up at least ten times more glutamate from its surroundings than amesenchymal stem cell.

According to some embodiments of the invention, the isolated human cellis not genetically manipulated.

According to some embodiments of the invention, the cell population isnon-genetically manipulated.

According to some embodiments of the invention, the isolated human cellfurther comprises an astrocytic structural phenotype.

According to some embodiments of the invention, the N % of the humancells comprise a structural phenotype.

According to some embodiments of the invention, the astrocyticstructural phenotype is expression of at least one astrocytic marker.

According to some embodiments of the invention, the isolated human cellfurther expresses at least one additional neurotrophic factor.

According to some embodiments of the invention, the at least oneadditional neurotrophic factor is selected from the group consisting ofglial derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3),neurotrophin-4/5, Neurturin (NTN), Persephin, artemin (ART), ciliaryneurotrophic factor (CNTF), insulin growth factor-I (IGF-1) andNeublastin.

According to some embodiments of the invention, the cell populationfurther expresses at least one additional neurotrophic factor.

According to some embodiments of the invention, the at least oneneurotrophic factor is GDNF.

According to some embodiments of the invention, the isolated human celldoes not secrete nerve growth factor (NGF).

According to some embodiments of the invention, the isolated human celltakes up at least ten times more glutamate from its surroundings than amesenchymal stem cell.

According to some embodiments of the invention, the mesenchymal stemcells comprise human mesenchymal stem cells and the platelet lysatecomprises human platelet lysate.

According to some embodiments of the invention, the medium is devoid ofxeno contaminants.

According to some embodiments of the invention, to duration of theincubating mesenchymal stem cells in a culture medium comprisingplatelet lysate is at least four weeks.

According to some embodiments of the invention, the culture medium isdevoid of serum.

According to some embodiments of the invention, a percentage of theplatelet lysate in the culture medium is about 5%.

According to some embodiments of the invention, a percentage of theplatelet lysate in the culture medium is about 10%.

According to some embodiments of the invention, the CNS disease ordisorder is a neurodegenerative disease or disorder.

According to some embodiments of the invention, the CNS disease ordisorder is selected from the group consisting of a motion disorder, adissociative disorder, a mood disorder, an affective disorder, anaddictive disorder and a convulsive disorder.

According to some embodiments of the invention, the neurodegenerativedisorder is selected from the group consisting of Parkinson's, multiplesclerosis, epilepsy, amyotrophic lateral sclerosis, stroke, autoimmuneencephalomyelitis, diabetic neuropathy, glaucomatous neuropathy,Alzheimer' s disease and Huntingdon' s disease.

According to some embodiments of the invention, the cells comprise anastrocytic phenotype.

According to some embodiments of the invention, the cells comprise anoligodendrocytic phenotype.

According to some embodiments of the invention, the cells secrete atleast one neurotransmitter.

According to some embodiments of the invention, the at least oneneurotransmitter is dopamine.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a graph illustrating the growth potential of MSC in mediumcontaining 5% Platelet lysate (PM) as compared to MSC grown in mediumcontaining 15% FCS (FCS). FIGS. 2A-2I are graphs illustrating theresults of FACS analyses of plastic adherent MSC grown in 5% plateletlysate for approximately 4 weeks. The cells were negative forHematopoetic surface markers: (CD3, T cell receptor; CD14monocyte/macrophages; CD19, B cell marker; CD34 hematopoieticprogenitors; CD45 pan-leukocyte marker, and HLA-DR) and stained positivefor mesenchymal surface markers CD73, CD105 and CD90.

FIGS. 3A-3J are photomicrographs illustrating that human bone marrowderived MSC express and secrete neurotrophic factors. MSC and NTF-SCwere stained with antibodies against neurotrophic factors includingBrain Derived Neurotrophic Factor (BNDF) (A-B), Glial-DerivedNeurotrophic factor (GDNF) (C-D) and Insulin-like growth factor 1 (IGF1)(E-F), and with antibodies against the astrocytic markers Glialfibrillary acidic protein (GFAP) (G-H) and Glutamine Synthetase (GS)(I-J). All secondary antibodies used were conjugated to Alexa-488(green). Nuclei were visualized with DAPI (blue). Scale bar=50 μm.

FIGS. 3K-3L are bar graphs illustrating the amount of neurotrophicfactors secreted by the NTF-SCs of the present invention. Neurotrophicfactor secretion was measured from human MSCs prior to and followinginduction of differentiation into NTF secreting cells, from 3 (BDNF) and5 (GDNF) different donors analysed by ELISA assay. * p<0.05 for the MSCvs the NTF secreting cells (Student's t test). For BDNF (FIG. 3K): Donor1: prior to differentiation=2666 pg/10⁶ cells, followingdifferentiation=9527 pg/10⁶ cells; Donor 2: prior to differentiation=520pg/10⁶ cells, following differentiation=6903 pg/10⁶ cells; Donor 3:prior to differentiation=463 pg/10⁶ cells, followingdifferentiation=4919 pg/10⁶ cells.

FIG. 4 is a graph illustrating that human MSC and NTF-SC mediaattenuated 6-OHDA induced neuroblastoma cells death. In the range of32-160 μM, 6-OHDA treatment resulted in a survival rate of less than 35%compared to untreated SH-SYSY neuroblastoma cells. Media from both celltypes of cells media attenuated the 6-OHDA induced cellular death in astatistically significant manner in comparison to neuroblastoma cellstreated with 6-OHDA only (p<0.05 for the range of 32-72 μM). A treatmentwith 160 μM of 6-OHDA resulted in a smaller but statisticallysignificant increase in cellular viability by the NTF-SC media only andnot by MSC media.

FIGS. 5A-5B are graphs illustrating that striatal transplantation ofhuman NTF-SC attenuate 6-OHDA-induced behavioral changes. (FIG. 5A) Inthe amphetamine induced rotations test only NTF-SC (n=20) treatment wasbeneficial compared to control (n=10) at two time points (day 28 and day42). No statistically significant difference was shown for the MSCtreated group (n=20) as compared to the control group or the NTF-SCtreated group. In contrast, for the NTF-SC groups, a marked decrease of25% and 45% after 14 and 28 days post transplantation was noted (FIG.5B) 6-OHDA induced a hypoactive motor behavior pattern in an open fieldtest at 7 days post treatment. NTF-SC treatment showed a positive effectin the voluntary mobility as compared to MSC- and PBS-treated groups(ANOVA, p=0.054).

FIGS. 6A-6E are illustrations and photographs illustrating themethodology of the stereological quantification of TH-positive striatalarea in the lesioned rats. As depicted in FIG. 6C, striatal coronalsections were made into 40 μm sections, and every 8^(th) section wasstained for thyrosine-hydroxylase (TH). Each section was captured by aX40 magnification and divided into 2-3 images from each side in asymmetrical fashion (FIGS. 6B, 6D). Using ImagePro software, a histogrambased cutoff was determined and a mask was created (FIGS. 6A, 6E). Thefinal quantification was performed by calculating the total red area inthe masked images.

FIGS. 7A-7E are photographs and graphs illustrating that treatment withhuman NTF-SC salvaged TH-positive striatal terminals damaged by 6-OHDA.(FIGS. 7A-7C) a macro-view of TH staining in striatum with arepresentative slice for each group of rats (A-PBS, B-MSC, C-NTF-SC).(FIG. 7D) Digital quantification of the TH-positive area in the wholelesioned striatum as a percent of the untreated striatum, demonstratingthe beneficial protective effect of NTF-SC treatment (n=4 from eachgroup,|—P<0.05 compared to PBS). (FIG. 7E) quantification of TH positivearea of the lesioned side compared to the untreated side—a segmentalcomparison. The whole striatum was divided into five segments fromanterior to posterior (each group represent approximately 1 mm thicknessof the striatum, *—p<0.05 compared to PBS).

FIGS. 8A-8G are images obtained following tracking of human NTF-SC byin-vivo MRI and corresponding histology. (FIG. 8A) T2* MRI scanconducted at 35 days post treatment of a control SPIOs-treated animal(without cells) demonstrating the SPIO injection site (blue circle) andthe 6-OHDA injection site in the striatum (red circle). No otherhypointense signals could be detected in the striatum. (FIG. 8B) Themigratory pathway as demonstrated by an axial T2* weighted image. Amarked hypointense signal is visible from the cell transplantation site(blue circle) to the 6-OHDA lesion site (red arrow). A good correlationto the Prussian blue staining was found (FIG. 8D) along the CC into theanterior striatum. The same trail (red arrow) can even be seen also inthe T2 weighted image (FIG. 8C) which is less sensitive to theinhomogeniety induced by the SPIOs. (FIG. 8D) Prussian blue stain todetect SPIOs-labeled cells in an axial section (50 days after treatmentin the animal that underwent MRI scan displayed in FIGS. 8B-8C)demonstrating a migratory path from the cell transplantation site (whitearrow), along the corpus callosum (CC, hollow black arrows) and into theanterior striatum (STR, LV-lateral ventricle, scale bar −500 μm). (FIGS.8E-8G) Immunostaining with anti-human nuclei antibody of the markedwhite boxes in FIG. 8D in adjacent sections (scale bar 100 μm).

FIG. 9 is a bar graph illustrating that induction of MSC into NTF-SCincreases glutamate uptake. [³H]-D-aspartate uptake (50 nM) was measuredin hMSC and in NTF-SC. Uptake was performed in the presence of Na⁺unless indicated otherwise. Na⁺ dependent uptake was calculated bysubtracting the results obtained from the Na free tests from the resultsof the control group. Competitive inhibition with D-methyl-aspartate (50nM) was performed in the presence of [³H]-D-aspartate at the sameconcentration as the other tests (50 nM). Inhibition with t-PDC, cellswere preincubated with t-PDC for 15 minutes. NTF-SC perform glutamateuptake significantly better then MSC (P<0.0001). Inhibitorssignificantly decrease the uptake of glutamate in NTF-SC as comparedwith NTF-SC control (competitive inhibition P<0.0005, t-PDC P<0.0001).

FIGS. 10A-10C are graphs and photomicrographs illustrating theexpression of GFAP in the differentiated cells of the present invention,as measured by real-time RT-PCR (FIG. 10A) and immunocytochemistry(FIGS. 10B-10C).

FIGS. 11A-11C are photomicrographs comparing the expression of S100 innon-differentiated (FIG. 11A) and differentiated (FIGS. 11B-11C) cellsof the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention relates to cells and populations thereof which canbe transplanted into a patient in order to treat a myriad ofneurodegenerative diseases.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Neurotrophic factors (NTFs) are secreted proteins that regulate thesurvival, functional maintenance and phenotypic development of neuronalcells. Alterations in NTF levels are involved in triggering programmedcell-death in neurons and thus contribute to the pathogenesis ofParkinson's disease and other neurodegenerative diseases.

One of the most potent NTF for dopaminergic neurons is called glial cellline-derived neurotrophic factor (GDNF). It is known to promote thesurvival of the dopaminergic neurons in the substantia nigra, promoteneurite outgrowth, increase cell body size and also raise levels of TH.Another potent NTF for dopaminergic neurons is brain derivedneurotrophic factor (BDNF). This NTF has potent effects on survival andmorphology of dopaminergic neurons and its loss has been postulated tocontribute to the death of these cells in Parkinson's disease (PD).

However, direct use of neurotrophic is prohibited as they do not passthe blood-brain barrier and do not distribute properly followingsystemic injection. Therefore, other strategies must be developed inorder to take advantage of their therapeutic properties.

Protocols for differentiating human mesenchymal stem cells intoneurotrophic factor secreting cells are known in the art—see for exampleWO 2006/134602.

Whilst searching for a way to increase the amount of neurotrophicfactors secreted by mesenchymal stem cells, the present inventors havefound that propagation of mesenchymal stem cells in platelet lysateprior to differentiation increased secretion of neurotrophic factorstherefrom.

The present inventors have shown that MSCs differentiated followingincubation in platelet lysate represent an astrocyte like shapeaccompanied by the presence of astrocyte markers. These cells were shownto express and secrete significant levels of GDNF and BDNF. Indeed,using immunocytochemistry procedures, the present inventors have shownthat approximately 90% of the cells generated express GDNF, BDNF, S100,GFAP and glutamine synthetase (FIGS. 3A-3J, 10A-10C and 11A-11C).

Following transplantation into the striatum of 6-OHDA lesioned rat (arodent model for Parkinson's) the cells survived for three months (asmeasured by MRI) and improved behavioral deficits examined by an openfield test, and apomorphine induced rotational behavior. In addition,the cells were shown to reduce dopamine depletion and causereinnervation of dopaminergic terminals. It was found that the efficacyof NTF-SC was superior to that of MSC in terms of behavioral,biochemical and histological indices. In addition, the present inventorsdemonstrated that the surviving cells migrated towards the lesion, andhad the most significant effect at the end of the migration trail.Further, the present inventors showed that MSCs differentiated accordingto the protocols of embodiments of the present invention are capable oftaking up glutamate.

Since the novel technology presented herein is clinically compatible andsafe, the present inventors propose that the transplantation of NTF-SCderived from autologous human MSC should become an important option intreatment of neurodegenerative disorders.

Thus, according to one aspect of the present invention there is provideda method of generating cells useful for treating a neurodegenerativedisorder, the method comprising:

-   -   (a) incubating mesenchymal stem cells in a culture medium        comprising platelet lysate to generate propagated mesenchymal        stem cells; and    -   (b) incubating the propagated mesenchymal stem cells in a        differentiating medium, thereby generating cells useful for        treating the neurodegenerative disorder.

The term “mesenchymal stem cell” or “MSC” is used interchangeably foradult cells which are not terminally differentiated, which can divide toyield cells that are either stem cells, or which, irreversiblydifferentiate to give rise to cells of a mesenchymal cell lineage. Themesenchymal stem cells of the present invention may be of a syngeneic orallogeneic source, although the first is preferred.

According to a preferred embodiment of this aspect of the presentinvention the mesenchymal stem cells are not genetically manipulated(i.e. transformed with an expression construct) to generate the cellsand cell populations described herein.

It will be appreciated that the cells of the present invention may bederived from any stem cell, although preferably not ES cells.

Mesenchymal stem cells may be isolated from various tissues includingbut not limited to bone marrow, peripheral blood, blood, placenta andadipose tissue. A method of isolating mesenchymal stem cells fromperipheral blood is described by Kassis et al [Bone Marrow Transplant.2006 May;37(10):967-76]. A method of isolating mesenchymal stem cellsfrom placental tissue is described by Zhang et al [Chinese MedicalJournal, 2004, 117 (6):882-887]. Methods of isolating and culturingadipose tissue, placental and cord blood mesenchymal stem cells aredescribed by Kern et al [Stem Cells, 2006;24:1294-1301].

According to a preferred embodiment of this aspect of the presentinvention, the mesenchymal stem cells are human.

Bone marrow can be isolated from the iliac crest of an individual byaspiration. Low-density BM mononuclear cells (BMMNC) may be separated bya FICOL-PAGUE density gradient. In order to obtain mesenchymal stemcells, a cell population comprising the mesenchymal stem cells (e.g.BMMNC) may be cultured in a proliferating medium capable of maintainingand/or expanding the cells in the presence of platelet lysate. Accordingto one embodiment the populations are plated on polystyrene plasticsurfaces (e.g. in a flask) and mesenchymal stem cells are isolated byremoving non-adherent cells. Alternatively mesenchymal stem cell may beisolated by FACS using mesenchymal stem cell markers.

Following isolation the cells are typically expanded by culturing in aproliferation medium capable of maintaining and/or expanding theisolated cells ex vivo in the presence of platelet lysate. Theproliferation medium may be DMEM, alpha-MEM or DMEM/F12.

It will be appreciated that preferably when the mesenchymal stem cellsare human, the platelet lysate is also obtained from human cells.

According to one embodiment, the medium is devoid of xeno contaminantsi.e. free of animal derived components.

An exemplary mesenchymal stem cell isolation and propagation protocol ispresented herein below.

Isolation of Human BM-MSC

Bone marrow samples (3-30 ml) were collected into EDTA containing tubesfrom the posterior iliac crest of adult human donors undergoing bonemarrow aspiration in the course of diagnostic procedures. Bone marrowaspirates were diluted 1:1 with HBSS and mononuclear cells wereseparated by density centrifugation (1000× G for 20 min), over UNI-SEPMAXI (Polysucrose—Sodium Metrizoate) containing tubes. The mononuclearcell fraction was collected and washed in HBSS. Cells were re-suspendedin Growth Medium containing 10% Platelet lysate (PM1), counted by theTrypan blue exclusion dye and seeded at a concentration of250,000-350,000 cells/cm² in 75 cm² tissue culture flasks. Flasks wereincubated in a 37° C. humidified incubator with 5% CO₂.

PM1 growth medium consisted of Dulbecco's Modified Eagle's Medium lowglucose (Sigma, Aldrich), supplemented with 0.05 mg/ml Gentamycin(Sigma, Aldrich), 2 IU/ml Heparin (TRIMA), 0.001% 2-mercaptoethanol(Sigma, Aldrich), 1% non-essential essential amino acid solution (Sigma,Aldrich) and 10% platelet lysate. 24 hrs later PM1 medium was aspiratedto remove non-adherent cells from the flask, adherent cells were washedgently with 10 ml of DMEM, and 10 ml of fresh PM1 were added to theflask. hMSC cells were allowed to proliferate for 12-18 days in PM1medium, which was replaced twice weekly. After 12-18 days or when theflask reached confluence. The cells were harvested by removing allgrowth medium and incubating in TrypLE™ solution (Invitrogen) for 5 minin a 37° C. incubator. Cells are then washed in DMEM, counted,resuspended in PM medium and seeded in CellStacks at a density of500-3000 cells/cm².

PM growth medium consists of Dulbecco's modified eagle's medium lowglucose supplemented with 0.05 mg/ml Gentamycin, 2 IU /ml Heparin and 5%platelet lysate. MSC cultures were passaged approximately every twoweeks by detachment of the sub-confluent cell layer with TrypLE™solution (Invitrogen). Experiments with the cells were performed after2-7 passages. Accordingly, the cells were passaged for a minimum of twoweeks in platelet lysate containing medium.

Platelet lysate may be prepared using any method known in the art. Anexemplary freeze-thaw protocol is provided herein below.

Preparation of Platelet Lysate

Platelet Rich Plasma (PRP) may be from Blood Bank donations determinedfree of infectious agents (i.e. HIV, HTLV, HCV, HBsAg). PRP containingbags were stored at −80° C. and thawed in a 37° C. water bath. Afterthawing, the Platelet Rich Plasma of multiple donors was pooled, mixedand centrifuged at 14000× G for 10 minutes to remove platelet particlesand membranes. The Platelet lysate supernatant was then collected andfrozen at −80° C. until use. The Platelet lysate was tested forEndotoxin, Haemoglobin, pH, Total protein, Albumin, Osmolality Sterilityand Mycoplasma.

Verification that the isolated (and optionally propagated) cellpopulation comprises mesenchymal stem cells may be effected byidentification of phenotypic and functional criteria. The phenotypiccriteria include the expression of specific surface antigens: CD73, CD90and CD105 (>=95% positive) and the absence (<2%) of (T-cells), CD14(Monocyte surface marker), CD19 (B cells), CD34 (Hematopoietic stemcells), CD45 (Hematopietic cells), and HLA-DR (Human Class IIHistocompatibility antigen). The surface expression of these cells maybe analyzed using methods known in the art—for example by FlowCytometry.

Exemplary antibodies that may be used to verify the presence ofmesenchymal stem cells include CD73 PE conjugated (BD Pharmingen), CD90PE-Cy5 conjugated (eBioscience) CD105 PE conjugated (Beckman Coulter)CD14 FITC conjugated (eBioscience) CD19 PE-Cy5 conjugated (eBioscience)CD34 FITC conjugated (Beckman Coulter), CD45 PE conjugated (eBioscience)and HLA-DR PE-Cy5 conjugated (BD Pharmingen).

Another method for verifying the presence of mesenchymal stem cells isby showing that the cells are capable of differentiating intomulti-lineages such as for example adipocytes, osteocytes andchondrocytes. This may be effected for example using Human MesenchymalStem Cell Functional Identification Kit (R&D Systems).

As mentioned, following propagation of mesenchymal stem cells in aplatelet lysate containing medium, the cells may be differentiated in adifferentiating medium to generating cells useful for treating aneurodegenerative disorder.

It will be appreciated that the components of the differentiating mediumare selected according to the cell phenotype required.

Thus according to one embodiment, the phenotype may be astrocyte-likecells.

As used herein the phrase “astrocyte-like cells” refers to cellscomprising at least one astrocytic phenotype which allows same to invivo mediate an astrocytic activity, i.e., support of neurons.

Such phenotypes are further described hereinbelow.

Differentiation to astrocyte-like cells can be effected by incubatingthe MSCs in differentiating media such as those described in U.S. Pat.No. 6,528,245 and by Sanchez-Ramos et al. (2000); Woodburry et al.(2000); Woodburry et al. (J. Neurisci. Res. 96:908-917, 2001); Black andWoodbury (Blood Cells Mol. Dis. 27:632-635, 2001); Deng et al. (2001),Kohyama et al. (2001), Reyes and Verfatile (Ann. N. Y. Acad. Sci.938:231-235, 2001) and Jiang et al. (Nature 418:47-49, 2002).

WO2006/134602, incorporated herein by reference, teaches differentiationprotocols for the generation of neurotrophic factor secreting cells.

According to one embodiment in order to generate astrocyte like cells,MSCs are initially incubated in a medium comprising epidermal growthfactor hEGF (e.g. 20 ng/ml) and/or basic fibroblast growth factor (e.g.20 ng/ml) in the presence or absence of N2 supplement (insulin,progesterone, putrescin, selenium and transferrin). Following this theBMScs may be differentiated in a second medium comprising plateletderived growth factor (e.g. 5 ng/ml) and human neuregulin 1-β1 (e.g. 50ng/ml). This “differentiating medium” may also include differentiatingagents such as IL-1β and/or dbcAMP.

According to one embodiment, the MSCs are incubated in eachdifferentiating medium for at least 24 hours. It will be appreciatedthat longer culturing times are contemplated, such as two days, threedays, four days or more.

According to another embodiment, the components of the differentiatingmedium are selected so as to generate cells comprising anoligodendrocyte phenotype. The differentiating media may be DMEM orDMEM/F12, or any other medium that supports neuronal growth. Accordingto a preferred embodiment of this aspect of the present invention, themedium is Neurobasal medium (e.g. Cat. No. 21103049, Invitrogen, Ca,U.S.A.). Table 1 herein below, summarizes various differentiationprotocols for the generation of oligodendrocytes.

TABLE 1 Stage Medium^(a) Days Control Regular growth medium: 13 α-MEMFCS 15% L-glutamine 2 mM Penicillin 100 U/ml Streptomycin 100 ug/mlNystatin 12.5 U/ml Protocol A Differentiating Neurobasal medium 13Medium (A) N2 supplement B27 supplement bFGF 10 ng/ml GGF 50 ng/mldb-cAMP 1 nM Protocol B Additional Neurobasal medium 5 Medium (B) PDGF20 ng/ml NT-3 10 ng/ml Il-1β 20 ng/ml Differentiating Neurobasal medium8 Medium (B) N2 supplement NT-3 10 ng/ml Il-1β 20 ng/ml Protocol CAdditional Neurobasal medium 5 Medium (C) TH 30 ng/ml (stock 20 ug/ml)RA 1 μM GGF 50 ng/ml Differentiating Neurobasal medium 8 Medium (C) TH30 ng/ml (stock 20 ug/ml) RA 1 μM NT-3 10 ng/ml Protocol D AdditionalNeurobasal medium 5 Medium (D) PDGF 20 ng/ml GGF 50 ng/mlDifferentiating Neurobasal medium 8 Medium (D) Shh 300 ng/ml NT-3 10ng/ml db-cAMP 1 nM Forskolin 5 μM

WO2007/066338, incorporated herein by reference, teaches differentiationprotocols for the generation of oligodendrocyte-like cells.

According to another embodiment, the components of the differentiatingmedium are selected so as to generate cells that secrete dopamine.

An exemplary method for differentiating mesenchymal stem cells intoneurotransmitter (e.g. dopamine) secreting cells is detailed in Table 2,herein below.

TABLE 2 Stage 1: DMEM/F12 (without HEPES); 2 mM L-glutamine; AdditionalSPN; 10 ng/ml human basic fibroblast growth factor differentiation(bFGF); 10 ng/ml EGF; *N2 supplement; 40 μM medium arachidonic acid;10-40 μM docosahexaenoic acid (24-72 hr) (DHA); 40 μM Vit-E; 10 ng/mlfibroblast growth factor 8 (FGF8); 200 ng/ml sonic hedgehog (Shh) Stage2: DMEM/F12; 2 mM L-glutamine; SPN; *N2 supplement; Dopaminergic 200 μMascorbic acid; 1 mM dibutyryl cyclic AMP; 0.5 differentiation mMisobutylmethlxanthine; 1 μM retinoic acid; 200 medium μM butylatedhydroxyanisole (BHA); human trans- (12-96 hr) forming growth factor β3(TGF- β3), 2 ng/ml; human galia-derived neurotrophic factor: (GDNF), 2ng/ml; human neurturin: (hNTN), 20 ng/ml; human brain- derivedneurotrophic factor: (BDNF), (10 ng/ml; human neurotrophin: (hNT-3), 20ng/ml; human interleukin-1β (hIL-1β), 100 pg/ml;

WO2004/046348, incorporated herein by reference, teaches differentiationprotocols for the generation of neuronal-like cells.

It will be appreciated that any of the differentiating media maycomprise other agents such as neurotrophic factors (e.g. BDNF, CNTF,GDNF, NTN, NT3 or LIF), hormones, growth factors (e.g. GGF2, TGF-β3,TGF-α, FGF-8 and bFGF), vitamins, hormones e.g., insulin, progesteroneand other factors such as sonic hedgehog, bone morphogenetic proteins,forskolin, retinoic acid, ascorbic acid, putrescin, selenium andtransferrin.

As mentioned, the present inventors showed that propagation ofmesenchymal stem cells in platelet lysate prior to incubation with adifferentiating agent which steers the mesenchymal stem cell towards anastrocytic phenotype (e.g. platelet derived growth factor (PDGF), humanneuregulin 1-β1, FGF2, EGF, N2, IBMX and cAMP), generated cells capableof secreting large amounts of neurotrophic factors.

Thus, according to another aspect of the present invention, there isprovided an isolated human cell comprising at least one mesenchymal stemcell phenotype and secreting brain-derived neurotrophic factor (BDNF),wherein a basal secretion of the BDNF is at least five times greaterthan a basal secretion of the BDNF in a mesenchymal stem cell.

The term “isolated” as used herein refers to a cell that has beenremoved from its in-vivo location (e.g. bone marrow, neural tissue).Preferably the isolated cell is substantially free from other substances(e.g., other cell types) that are present in its in-vivo location.

The mesenchymal stem cell phenotypes which are comprised in the cells ofthe present invention are typically structural. For example, the cellsof the present invention may show a morphology similar to that ofmesenchymal stem cells (a spindle-like morphology). Alternatively oradditionally the cells of the present invention may express a marker(e.g. surface marker) typical to mesenchymal stem cells but atypical tonative astrocytic cells. Examples of mesenchymal stem cell surfacemarkers include but are not limited to CD105+, CD29+, CD44+, CD90+,CD34−, CD45−, CD19−, CD5−, CD20−, CD11B− and FMC7−. Other mesenchymalstem cell markers include but are not limited to tyrosine hydroxylase,nestin and H-NF.

As mentioned, the basal secretion of BDNF from cells according to thisaspect of the present invention is at least five times greater than abasal secretion of the BDNF in a non-differentiated mesenchymal stemcell.

It will be appreciated that the basal secretion of BDNF may be evenhigher, such as at least six times greater, at least seven timesgreater, at least eight times greater, at least nine times greater oreven at least ten times greater.

According to one embodiment, at least 50%, at least 60%, at least 70%,at least 80%, at least 90% or more of a population of the differentiatedcells of the present invention express BDNF.

As used herein the term “basal secretion” refers to a secretion whichdoes not involve addition of stimulants. The non-differentiatedmesenchymal stem cell is typically obtained from the same source as thedifferentiated mesenchymal stem cell and is identical thereto apart fromhaving been differentiated according to the protocols described herein.Thus typically, the non-differentiated mesenchymal stem cell is in anidentical medium to the differentiated mesenchymal stem cells butwithout the addition of differentiating agents.

According to one embodiment the cells of the present invention arecapable of taking up glutamate from their surrounding milieu (e.g.culture medium). For example, the cells of the present invention may becapable of taking up at least 10 times, at least 20 times, at least 30times, at least 40 times or even at least 50 times more glutamate fromtheir surroundings than a non-differentiated mesenchymal stem cell.

The cells of the present invention may also comprise an astrocyticphenotype.

As used herein, the phrase “astrocytic phenotype” refers to a structuraland/or functional parameter typical (e.g. unique) to an astrocyte whichmay be used to distinguish between the differentiated MSCs of thepresent invention and non-differentiated MSCs. The astrocytic phenotypemay comprise a single or a number of features which may be used todistinguish between the differentiated MSCs of the present invention andnon-differentiated MSCs.

It will be appreciated that the functional parameters may overlap withthe structural parameter e.g., presence of secretory vesicles.

According to one embodiment the functional astrocytic phenotypecomprises the ability to express an additional neurotrophic factor.

As used herein the term “express” refers to the synthesis and/orsecretion of the above-mentioned neurotrophic factor.

As used herein, the phrase “neurotrophic factor” refers to a cell factorthat acts on the cerebral nervous system comprising growth,differentiation, functional maintenance and/or survival effects onneurons. Examples of neurotrophic factors include, but are not limitedto, glial derived neurotrophic factor (GDNF), GenBank accession nos.L19063, L15306; brain-derived neurotrophic factor (BDNF), GenBankaccession no CAA62632; neurotrophin-3 (NT-3), GenBank Accession No.M37763; neurotrophin-4/5; Neurturin (NTN), GenBank Accession No.NP_004549; Neurotrophin-4, GenBank Accession No. M86528; Persephin,GenBank accession no. AAC39640; brain derived neurotrophic factor,(BDNF), GenBank accession no. CAA42761; artemin (ART), GenBank accessionno. AAD13110; ciliary neurotrophic factor (CNTF), GenBank accession no.NP_000605; insulin growth factor-I (IGF-1), GenBank accession no.NP_000609; and Neublastin GenBank accession no. AAD21075.

According to one embodiment, at least 50%, at least 60%, at least 70%,at least 80%, at least 90% or more of a population of the differentiatedcells of the present invention express GDNF.

Typically the cells of the present invention do not secrete nerve growthfactor (NGF).

Examples of structural astrocytic phenotypes include a cell size, a cellshape, an organelle size and an organelle number. Thus, astrocyticstructural phenotypes include a round nucleus, a “star shaped” body andmany long processes that end as vascular foot plates on the small bloodvessels of the CNS. Further examples of structural astrocytic phenotypesmay be found in the following materials: Reynolds and Weiss, Science(1992) 255:1707-1710; Reynolds, Tetzlaff, and Weiss, J. Neurosci (1992)12:4565-4574; and Kandel, et al., Principles of Neuroscience, third ed.(1991), Appleton & Lange, Norwalk, Conn. These structural phenotypes maybe analyzed using microscopic techniques (e.g. scanning electromicroscopy). Antibodies or dyes may be used to highlight distinguishingfeatures in order to aid in the analysis.

A structural astrocytic phenotype may also comprise expression of anastrocyte marker.

As used herein the phrase “astrocyte marker” refers to a polypeptidewhich is either selectively or non-selectively expressed in anastrocyte. The astrocyte marker may be expressed on the cell surface orinternally. Examples of astrocyte markers include S100 beta, glialfibrillary acidic protein (GFAP), glutamine sythetase (GS), GLAST andGLT1.

According to one embodiment, at least 50%, at least 60%, at least 70%,at least 80%, at least 90% or more of a population of the differentiatedcells of the present invention express at least one or more astrocytemarkers including, but not limited to S100 beta, GFAP and GS.

It will be appreciated that cell populations obtained according to themethods describe herein are typically non-homogeneous.

Thus according to another aspect of the present invention there isprovided an isolated cell population comprising human cells wherein:

-   -   (i) at least N % of the human cells secreting brain-derived        neurotrophic factor (BDNF), wherein a basal secretion of the        BDNF is at least five times greater than a basal secretion of        the BDNF in a mesenchymal stem cell;    -   (ii) at least M % of the human cells comprise at least one        mesenchymal stem cell phenotype; and    -   (iii) at least one of the human cells secretes the BDNF and the        at least one mesenchymal stem cell phenotype;    -   where M and N are each independently selected between 1 and 99.

M % may be any percent from 1% to 99% e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% and 99%.

N % may be any percent from 1% to 99% e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% and 99%.

The percentage of cells which secrete BDNF may be raised or loweredaccording to the intended needs. This may be effected by FACS using anantibody specific for an astrocyte cell marker. Examples of suchastrocytic markers are described hereinabove. If the cell marker is aninternal marker, preferably the FACS analysis comprises antibodies orfragments thereof which may easily penetrate a cell and may easily bewashed out of the cell following detection. The FACS process may berepeated a number of times using the same or different markers dependingon the degree of enrichment and the cell phenotype required as the endproduct.

According to another embodiment of this aspect of the present inventionthe cell populations may be enriched for cells comprising both anastrocytic phenotype and a mesenchymal stem cell phenotype such that ahomogeneous population of cells are generated.

Once differentiated and optionally isolated, the cells may be tested (inculture) for their ability to secrete a BDNF. An exemplary method foranalyzing secretion of BDNF (and other neurotrophic factors (NTFs) isdescribed herein below.

ELISA for Secreted NTFs

For analysis of secreted NTFs, supernatant is collected from cultures ofMSCs or of NTF-secreting cells at the end of the differentiationprocedure described above, and cells are harvested and counted. Theamount of NTFs such as Glial Derived Neurothrophic Factor, (GDNF) orBrain Derived Neurothrophic Factor (BDNF) in the cell's culturesupernatants was quantified by using a GDNF or BDNF ELISA assay (GDNFDuoSet DY212; BDNF DuoSet DY248; R&D Systems) according to themanufacture's protocol.

As mentioned according to the phenotype, the cells and cell populationsof the present invention may be used to treat a particular disease ordisorder. The cell populations may be used directly followingdifferentiation or may be enriched for a particular phenotype asdescribed hereinabove. As summarized in Table 3 hereinbelow, certainneurotrophic factors or set of neurotrophic factors have been shown tobe particularly beneficial for treating a particular disease. Forexample, cells of the present invention which secrete BDNF and GDNFwould be particularly suitable for treating Parkinson's.

TABLE 3 Disease Astrocytic phenotype REF Parkinsons BDNF, FGF, GDNFWalker D G, et al. Brain Res 1998; 794: 181-7. Lorigados L, et al. RevNeurol 1998; 26: 744-8. Mogi M, et al. Neurosci Lett 1994; 180: 147-50.Howells D W, et al. Exp Neurol 2000; 166: 127-35. Beck K D, et al.Nature 1995; 373: 339-41. Tomac A, et al. Nature 1995; 373: 335-9. GashD M, et al. Nature 1996; 380: 252-5. Choi-Lundberg D L, Science 1997;275: 838-41. Bozzi Y, Borrelli E. Eur J Neurosci 1999; 11: 1275-84.Chauhan N B, et al Soc Neurosci Abstr 1998; 24: 1465. Chauhan N B, etal, Neurology 1999; 52: A212-213. Epilepsy BDNF, NT-3, glutamate G. W.Mathern, Mol. Chem. Neuropathol. 30 1-2 (1997), transporter pp. 53-76.Lucia Tapia-Arancibia et al. Frontiers in Neuroendocrinology 2004 July;25(2): 77-107. RYUTA KOYAMA and YUJI IKEGAYA; NEUROSCIENCE UPDATE 2005August; 11(4): 282-7. Gerald Seifert, et al., Nature ReviewsNeuroscience 7, 194-206 (March 2006). ALS NT3, IGF1, BDNF, Luis H. Etal., Brain Research Reviews 2004 glutamate transporter December;47(1-3): 263-74. Bradley W G. Ann Neurol 1995; 38: 971. Haase G, et al.Nat Med 1997; 3: 429-36. Arakawa Y, J Neurosci 1990; 10: 3507-15. Drugand alcohol GDNF Ron D, Janak P H. Rev Neurosci. 2005; 16(4): 277-85.addiction Brain injury Ability of cells to Nancy Rothwell; Brain,Behavior, and Immunity. 2003 respond to IL-1 June; 17(3): 152-7.Alzheimers BDNF Crutcher K A, et al. J Neurosci 1993; 6: 2540-50. ScottS A, et al. Nerve growth factor in Alzheimer's disease: increased levelsthroughout the brain coupled with declines in nucleus basalis. JNeurosci 1995; 15: 6213-21. Peng S, et al. J Neuropathol Exp Neurol2004; 63: 641-9. Murer M G, et al. Neuroscience 1999; 88: 1015-32.Huntingdon's BDNF, NT-3, or NT-4/5 Martinez-Serrano A, Bjorklund A.Trends Neurosci 1997; 20: 530-8. Perez-Navarro E, et al. J Neurochem2000; 75: 2190-9. Perez-Navarro E, et al. Neuroscience 1999; 91:1257-64. Schizophrenia NT-3, BDNF Gal Shoval, Abraham Weizmana; EurNeuropsychopharmacol. 2005 May; 15(3): 319-29. Levi-Montalcini, R.,1987. Biosci. Rep. 7, 681-699. Hattori, M., Nanko, S., 1995. Biochem.Biophys. Res. Commun. 209, 513-518. Virgos, C., 2001, Schizophr. Res.49, 65-71. Optic nerve CNTF Paul A. Sieving, et al., Proc Natl Acad SciUSA. 2006 Mar. 7; 103(10): 3896-901. Stroke FGF, BDNF Wu D; Neuro Rx.2005 January; 2(1): 120-8.

It has been proposed that astrocyte cells may reduce the oxidativestress in neurons by metabolizing dopamine, as they express monoamineoxidase-B and catechol-O-methyl-transferase. Additionally, it has beenproposed that astrocyte cells may be capable of preventing NO generatedneurotoxicity by a glutathione dependent mechanism (Chen et al. 2004,Curr Drug Targets. 2005 Nov;6(7):821-33). Accordingly, cells of thepresent invention which comprise a scavenging function and/or expressdopamine metabolizing enzymes may also be suitable for treatingParkinson's.

Owing to insufficient clearance or decrease of the glutamatetransporters glutamate excitotoxicity has been suggested as a causativefactor for ALS [Bendotti 2001, et al, J Neurochem, 79(4):737-746, 2001].Thus cells of the present invention which show an elevated glutamatetransporter activity may also be suitable for treating ALS.

It will be appreciated that cells capable of secreting neurotransmittersuch as dopamine would be particularly suitable for treating Parkinson'sdisease.

Further, cells comprising an oligodendrocyte phenotype would beparticularly suitable for diseases and conditions of the nervous systemthat result from the deterioration of, or damage to, the myelinsheathing generated by myelin producing cells are numerous. Myelin maybe lost as a primary event due to direct damage to the myelin or as asecondary event as a result of damage to axons and neurons. Primaryevents include neurodegenerative diseases such as multiple sclerosis(MS), human immunodeficiency MS-associated myelopathy, transversemyelopathy/myelitis, progressive multi focal leukoencepholopathy,central pontine myelinolysis and lesions to the myelin sheathing (asdescribed below for secondary events). Secondary events include a greatvariety of lesions to the axons or neurons caused by physical injury inthe brain or spinal cord, ischemia diseases, malignant diseases,infectious diseases (such has HIV, Lyme disease, tuberculosis, syphilis,or herpes), degenerative diseases (such as Parkinson's, Alzheimer's,Huntington's, ALS, optic neuritis, postinfectious encephalomyelitis,adrenoleukodystrophy and adrenomyeloneuropathy), schizophrenia,nutritional diseases/disorders (such as folic acid and Vitamin B12deficiency, Wemicke disease), systemic diseases (such as diabetes,systemic lupus erthematosis, carcinoma), and toxic substances (such asalcohol, lead, ethidium bromide); and iatrogenic processes such as druginteractions, radiation treatment or neurosurgery.

Thus, according to another aspect of the present invention there isprovided a method of treating a CNS disease or disorder.

As used herein, the phrase “CNS disease” refers to any disorder, diseaseor condition of the central nervous system which may be treated with thecells of the present invention.

Accordingly, these cells can be used for preparing a medicament(interchangeably referred to as pharmaceutical composition), wherebysuch a medicament is formulated for treating a CNS disease or disorder.

Representative examples of CNS diseases or disorders that can bebeneficially treated with the cells described herein include, but arenot limited to, a pain disorder, a motion disorder, a dissociativedisorder, a mood disorder, an affective disorder, a neurodegenerativedisease or disorder and a convulsive disorder.

More specific examples of such conditions include, but are not limitedto, Parkinson's, ALS, Multiple Sclerosis, Huntingdon's disease,autoimmune encephalomyelitis, diabetic neuropathy, glaucomatousneuropathy, macular degeneration, action tremors and tardive dyskinesia,panic, anxiety, depression, alcoholism, insomnia, manic behavior,Alzheimer's and epilepsy.

In any of the methods described herein the cells may be obtained fromany autologous or non-autologous (i.e., allogeneic or xenogeneic) humandonor. For example, cells may be isolated from a human cadaver or adonor subject.

The cells of the present invention can be administered to the treatedindividual using a variety of transplantation approaches, the nature ofwhich depends on the site of implantation.

The term or phrase “transplantation”, “cell replacement” or “grafting”are used interchangeably herein and refer to the introduction of thecells of the present invention to target tissue. The cells can bederived from the recipient or from an allogeneic or xenogeneic donor.

The cells can be grafted into the central nervous system or into theventricular cavities or subdurally onto the surface of a host brain.Conditions for successful transplantation include: (i) viability of theimplant; (ii) retention of the graft at the site of transplantation; and(iii) minimum amount of pathological reaction at the site oftransplantation. Methods for transplanting various nerve tissues, forexample embryonic brain tissue, into host brains have been described in:“Neural grafting in the mammalian CNS”, Bjorklund and Stenevi, eds.(1985); Freed et al., 2001; Olanow et al., 2003). These proceduresinclude intraparenchymal transplantation, i.e. within the host brain (ascompared to outside the brain or extraparenchymal transplantation)achieved by injection or deposition of tissue within the host brain soas to be opposed to the brain parenchyma at the time of transplantation.

Intraparenchymal transplantation can be effected using two approaches:(i) injection of cells into the host brain parenchyma or (ii) preparinga cavity by surgical means to expose the host brain parenchyma and thendepositing the graft into the cavity. Both methods provide parenchymaldeposition between the graft and host brain tissue at the time ofgrafting, and both facilitate anatomical integration between the graftand host brain tissue. This is of importance if it is required that thegraft becomes an integral part of the host brain and survives for thelife of the host.

Alternatively, the graft may be placed in a ventricle, e.g. a cerebralventricle or subdurally, i.e. on the surface of the host brain where itis separated from the host brain parenchyma by the intervening pia materor arachnoid and pia mater. Grafting to the ventricle may beaccomplished by injection of the donor cells or by growing the cells ina substrate such as 3% collagen to form a plug of solid tissue which maythen be implanted into the ventricle to prevent dislocation of thegraft. For subdural grafting, the cells may be injected around thesurface of the brain after making a slit in the dura. Injections intoselected regions of the host brain may be made by drilling a hole andpiercing the dura to permit the needle of a microsyringe to be inserted.The microsyringe is preferably mounted in a stereotaxic frame and threedimensional stereotaxic coordinates are selected for placing the needleinto the desired location of the brain or spinal cord. The cells mayalso be introduced into the putamen, nucleus basalis, hippocampuscortex, striatum, substantia nigra or caudate regions of the brain, aswell as the spinal cord.

The cells may also be transplanted to a healthy region of the tissue. Insome cases the exact location of the damaged tissue area may be unknownand the cells may be inadvertently transplanted to a healthy region. Inother cases, it may be preferable to administer the cells to a healthyregion, thereby avoiding any further damage to that region. Whatever thecase, following transplantation, the cells preferably migrate to thedamaged area.

For transplanting, the cell suspension is drawn up into the syringe andadministered to anesthetized transplantation recipients. Multipleinjections may be made using this procedure.

The cellular suspension procedure thus permits grafting of the cells toany predetermined site in the brain or spinal cord, is relativelynon-traumatic, allows multiple grafting simultaneously in severaldifferent sites or the same site using the same cell suspension, andpermits mixtures of cells from different anatomical regions. Multiplegrafts may consist of a mixture of cell types, and/or a mixture oftransgenes inserted into the cells. Preferably from approximately 10⁴ toapproximately 10⁸ cells are introduced per graft.

For transplantation into cavities, which may be preferred for spinalcord grafting, tissue is removed from regions close to the externalsurface of the central nerve system (CNS) to form a transplantationcavity, for example as described by Stenevi et al. (Brain Res.114:1-20., 1976), by removing bone overlying the brain and stoppingbleeding with a material such a gelfoam. Suction may be used to createthe cavity. The graft is then placed in the cavity. More than onetransplant may be placed in the same cavity using injection of cells orsolid tissue implants. Preferably, the site of implantation is dictatedby the CNS disorder being treated and the astrocytic phenotype comprisedin the cell (e.g. particular neurotrophic factor being secreted) by thecells of the present invention. For example, cells secreting GDNF arepreferably implanted in the substantia nigra of a Parkinson's patient.

Since non-autologous cells are likely to induce an immune reaction whenadministered to the body several approaches have been developed toreduce the likelihood of rejection of non-autologous cells. Theseinclude either suppressing the recipient immune system or encapsulatingthe non-autologous cells in immunoisolating, semipermeable membranesbefore transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu MZ, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesTechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002;13:783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, abiological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

In any of the methods described herein, the cells can be administeredeither per se or, preferably as a part of a pharmaceutical compositionthat further comprises a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the chemical conjugates described herein, with otherchemical components such as pharmaceutically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to a subject.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

According to a preferred embodiment of the present invention, thepharmaceutical carrier is an aqueous solution of saline.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration include direct administration into thetissue or organ of interest. Thus, for example the cells may beadministered directly into the brain as described hereinabove ordirectly into the muscle as described in Example 3 hereinbelow.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. Preferably, a dose is formulated in ananimal model to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. For example, 6-OHDA-lesionedmice may be used as animal models of Parkinson's. In addition, asunflower test may be used to test improvement in delicate motorfunction by challenging the animals to open sunflowers seeds during aparticular time period.

Transgenic mice may be used as a model for Huntingdon's disease whichcomprise increased numbers of CAG repeats have intranuclear inclusionsof huntingtin and ubiquitin in neurons of the striatum and cerebralcortex but not in the brain stem, thalamus, or spinal cord, matchingclosely the sites of neuronal cell loss in the disease.

Transgenic mice may be used as a model for ALS disease which compriseSOD-1 mutations.

The septohippocampal pathway, transected unilaterally by cutting thefimbria, mimics the cholinergic deficit of the septohippocampal pathwayloss in Alzheimers disease. Accordingly animal models comprising thislesion may be used to test the cells of the present invention fortreating Alzheimers.

Survival and rotational behavior (e.g. on a rotarod) of the animals maybe analyzed following administration of the cells of the presentinvention.

The data obtained from these in vitro and cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition, (see e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1). For example,Parkinson's patient can be monitored symptomatically for improved motorfunctions indicating positive response to treatment.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Dosage amount and interval may be adjusted individually to levels of theactive ingredient which are sufficient to effectively regulate theneurotransmitter synthesis by the implanted cells. Dosages necessary toachieve the desired effect will depend on individual characteristics androute of administration. Detection assays can be used to determineplasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks ordiminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the individual being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc. The dosage and timing of administration willbe responsive to a careful and continuous monitoring of the individualchanging condition. For example, a treated Parkinson's patient will beadministered with an amount of cells which is sufficient to alleviatethe symptoms of the disease, based on the monitoring indications.

The cells of the present invention may be co-administered withtherapeutic agents useful in treating neurodegenerative disorders, suchas gangliosides; antibiotics, neurotransmitters, neurohormones, toxins,neurite promoting molecules; and antimetabolites and precursors ofneurotransmitter molecules such as L-DOPA. Additionally, the cells ofthe present invention may be co-administered with other cells capable ofsynthesizing a neurotransmitter. Such cells are described in U.S. Pat.Appl. No. 20050265983 to the present inventors.

Following transplantation, the cells of the present invention preferablysurvive in the diseased area for a period of time (e.g. at least 3months), such that a therapeutic effect is observed.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984); “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996) and Parfitt et al. (1987). Bonehistomorphometry: standardization of nomenclature, symbols, and units.Report of the ASBMR Histomorphometry Nomenclature Committee. J BoneMiner Res 2 (6), 595-610; all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Generation and Characterization ofNeurotrophic-Factor-Secreting Cells (NTF-SCs)

Materials and Methods

Preparation of Platelet Lysate

Platelet lysate was prepared using a freeze-thaw protocol. Platelet RichPlasma (PRP) were obtained from Blood Bank donations determined free ofinfectious agents (i.e. HIV, HTLV, HCV, HBsAg). PRP containing bags werestored at −80° C. and thawed in a 37° C. water bath. After thawing thePlatelet Rich Plasma of multiple donors was pooled, mixed andcentrifuged at 14000× G for 10 minutes to remove platelet particles andmembranes. The Platelet lysate supernatant was then collected and frozenat −80° C. until use. The Platelet lysate was tested for endotoxin,haemoglobin, pH, total protein, albumin, osmolality, sterility andmycoplasma.

Isolation and Proliferation of Human MSC:

Adult human bone marrow samples were collected from the posterior iliaccrest of adult human donors, undergoing bone marrow aspiration, afterobtaining informed consent. Bone marrow aspirates were diluted 1:1 withHanks' Balanced Salt Solution (HBSS, Biological Industries, Beit-Haemek,Israel) and mononuclear cells were separated by density centrifugation,over UNI-SEP_(maxI)/UNI-SEP+(Polysucrose—Sodium Metrizoate, NovaMed,Jerusalem, Israel) containing tubes. The mononuclear cell fraction wascollected, washed in HBSS and centrifuged. Cells were re-suspended inGrowth Medium 1, counted and seeded at a concentration of250,000-350,000 cells/cm² in 75 cm² tissue culture flasks (Corning, NY,USA). Flasks were incubated in a 37° C. humidified incubator with 5%CO₂.

Growth medium 1 consisted of Dulbecco's Modified Eagle's Medium (DMEM,Biological Industries), supplemented with 100 μg/ml streptomycin, 100U/ml penicillin, 12.5 units/ml nystatin (SPN, Biological industries), 2mM L-Glutamine (Biological industries), 2 IU/ml Heparin (Trima, KibutzMaabarot, Israel), 0.001% 2-mercaptoethanol (Sigma-Aldrich, St. Louis,Mo., USA), 1% MEM non-essential amino acid solution (BiologicalIndustries) and 10% Platelet lysate. Platelet lysate was processed fromfrozen-thawed human platelet rich plasma (PRP) as described hereinabove. 24 hrs later, Growth medium 1 was aspirated to removenon-adherent cells from the flask. Human MSC were allowed to proliferatein Growth medium 1, which was replaced twice weekly. After 12-18 daysthe cells were trypsinized (trypsin from Biological Industries),centrifuged, counted, resuspended in Growth medium 2 and seeded inCellStacks (Corning) at a density of 500-3000 cells/cm². Growth medium 2consists of DMEM supplemented with SPN, glutamine and heparin as inGrowth medium 1 and 5% PRP. MSC cultures were passaged approximatelyevery two weeks. Experiments on the cells were performed after 2-7passages.

Induction of Human MSC into NTF-SC

Human MSC (12,000 cells/cm²) were first placed in DMEM supplemented withSPN, 2 mM L-Glutamine (Biological industries), 20 ng/ml human epidermalgrowth factor (hEGF), 20 ng/ml human basic fibroblast growth factor(hbFGF) (R&D Systems) and N2 supplement (Invitrogen). After 72 hours,the medium was replaced with DMEM supplemented with 1 mM dibutyrylcyclic AMP (dbcAMP), 0.5 mM isobutylmethylxanthine (IBMX)(Sigma-Aldrich), 5 ng/ml human platelet derived growth factor (PDGF), 50ng/ml human neuregulin 1-β1/HRG1-β1 EGF domain and 20 ng/ml hbFGF (allfrom R&D Systems) for 3 more days.

Immunocytochemistry

Human MSC and NTF-SC were fixed with 4% paraformaldehyde and stainedwith rabbit anti-glial fibrillary acidic protein (GFAP;1:200, DAKO),rabbit anti glutamine synthetase (GS; 1:100; Sigma), rabbit anti-GDNF(1:100, Santa Cruz), rabbit anti-BDNF (1:100, Santa Cruz), rabbit antiIGF-1 (1:100, Santa Cruz). Secondary antibodies were goat anti-rabbitAlexa-488 (1:200, Molecular Probes). For GDNF staining, secondaryantibodies were biotinylated goat anti-rabbit (1:200; JacksonLaboratories) and streptavidine-Alexa-488 (1:200, Molecular Probes).Nuclear DNA was stained by 4,6-diamino-2-phenylindole (DAPI) (1:1000,Sigma).

In-Vitro Neuroprotection Assay

Neuroblastoma cell line SH-SYSY cells (ATCC, Manassas, Va., USA) weregrown in basal media consisting of DMEM with 10% fetal calf serum (FCS),2 mM L-glutamine, and SPN (Biological industries). The SH-SYSY cellswere plated in 96-well plates. Each well was applied with either humanMSC or NTF-SC conditioned media or with serum free medium (DMEM,Glutamine and SPN), and immediately exposed to oxidative insult by6-OHDA (Sigma-Aldrich) for 24 hours. Cell viability after treatments wasanalyzed by adding 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution to each well followed byincubation at 37° C. for 3 hours. Absorbance was determined at 564 nm ina microplate reader. Cell viability was evaluated in sextuplets for eachtreatment and compared to the serum free treated cells.

ELISA Based Measurements of NTFs Secretion

At the end of the NTF-SC induction process, the cell culture supernatantwas measured for human GDNF and BDNF concentrations by a sandwich ELISAprocedure according to the manufacturer's instructions (DuoSet, R&DSystem for human BDNF and GDNF). The absorbance at 450 nm and 570 nm wasrecorded on a Microplate Reader (Labsystems Multiscan MS). The resultswere calculated for one million cells.

Results

Analysis of MSC in Medium Containing 5% Platelet Lysate

The growth potential of MSC in medium containing 5% Platelet lysate (PM)as compared to MSC grown in medium containing 15% FCS (FCS) isdemonstrated in FIG. 1.

FACS analyses of plastic adherent MSC grown in 5% platelet lysate forapproximately 4 weeks is demonstrated in FIGS. 2A-2I. The cells werenegative for Hematopoetic surface markers: (CD3, T cell receptor; CD14monocyte/macrophages; CD19, B cell marker; CD34 hematopoieticprogenitors; CD45 pan-leukocyte marker, and HLA-DR) and stained positivefor Mesenchymal surface markers CD73, CD105 and CD90.

Induced Human NTF-SC Express Neurotrophic Factors

In-vitro analysis of the NTF-SC revealed their NTF expression profile.They expressed astrocyte markers such as GFAP and GS. Moreover, TheNTF-SC highly expressed the GDNF, BDNF and IGF-1 proteins as indicatedby immunocytochemistry (FIGS. 3A-3J). ELISA analysis showed that thedifferentiated NTF-SC secrete the neurotrophic factors into the culturesupernatant. While untreated MSC secreted low levels of BDNF and GDNF(1216±725 pg/10⁶ cells and 337±27 pg/10⁶ cells, respectively), after sixdays of induction the NTF-SC secreted over five times more BDNF(7117±1335 pg/10⁶ cells), and over twice the amount of GDNF (787±206pg/10⁶ cells) (FIGS. 3K-3L).

NGF was found to be negative when tested in supernatants of 7 differentMSCs donors propagated in PM and in 6 different supernatants ofNTF-secreting cells, using the ELISA kit to Human bNGF (DuoSet R&D,limit of Detection—2 pg/ml, linear between 4 and 250 pg/ml).

Conditioned Media from Human NTF-SC Protect Neuroblastoma Cell Lineagainst 6-OHDA Toxicity:

The protective effect of the induced MSC in vitro, was examined using aneuroblastoma cell line, SH-SY5Y, exposed to increasing doses of 6-OHDA(32-160 μM). For the neuroprotection assay, human MSCs were induced,following which the induction medium was removed. The induced cells werethen incubated with fresh serum free medium for an additional 24 hoursto allow secretion of NTFs. For controls, supernatants of untreated MSCincubated in serum free medium for 24 h were used. The SH-SY5Y cellswere incubated with the supernatants and one hour later, 6-OHDAneurotoxin was added to the cultures. Cell viability was measured 24hours later, using MTT. A consistent reduction in viability of theSH-SY5Y cells incubated with DMEM and 32, 48 and 72 μM of 6-OHDA(31.7±6.9%, 10.1±0.2% and 12.3±0.4%, respectively, FIG. 4) was noted. Incontrast, SH-SY5Y cells that were incubated with supernatants, collectedfrom untreated MSC or NTF-SC media, demonstrated a statisticallysignificant higher percentage of viability in the presence of 32, 48 and72 μM 6-OHDA. The NTF-SC media demonstrated an added protective valueupon the survival of the SH-SY5Y cells, although no statisticallysignificant difference was demonstrated between the induced NTF-SC mediatreatment and the untreated MSC media treatment (90.2±7.4%, 73.2±9.6%and 55.4±15.9%, respectively for the MSC group, p<0.05, and 92.4±5.0%,93.4±12.9% and 80.3±12% for the NTF-SC group, p<0.005, FIG. 4). At 160μM 6-OHDA, cell viability dropped to 15.0±0.6% in the control group andsimilarly in the MSC group (11.7±0.2%), while the NTF-SC media remainedslightly beneficial (17.9±0.6%, P<0.05 compared to both groups).

Example 2 In-Vivo Studies with the NTF-SCs of the Present Invention

Materials and Methods

6-OHDA Induced Striatal Lesion

A total of 63 male Sprague-Dawley rats weighting 260-300 gr (Harlan,Jerusalem, Israel) were used in this experiment. They were placed under12 hours light/12 hours dark conditions and grown in individualventilated cages (IVC) with ad libitum access to food and water.

7 μg/2.5 μl/site of 6-hydroxy dopamine (6-OHDA, Sigma-Aldrich) wasinjected into two sites (total of 14 μg 6-OHDA) in the right striatumaccording to the rat brain atlas (43) in 56 animals. Under chloralhydrate anesthesia the rats were placed in a digital stereotactic frame(Stoelting, Wood Dale, Ill., USA) and 6-OHDA was injected to thefollowing coordinates (relative to the bregma and dura): AP +0.5 ML 2.5DV −6.0 & AP −0.5 ML 4.2 DV −6.0 at a rate of 1 μl/minute using aHamilton 701 N syringe. The inserted needle was withdrawn from eachlocation after 5 minutes.

Stem Cell Transplantation

Human MSCs grown in serum free conditions for 6 days or induced NTF-SC,were used. On treatment day, the cells were trypsinized, washed withphosphate buffered saline (PBS) and counted. Two concentrations of cellswere injected (50,000 cells/μl or 150,000 cells/μl in PBS). A total of150,000 or 450,000 cells/3 μl were injected per site at a rate of 1μl/minute, and cells were transplanted into two sites along the same DVaxis: AP −1.8, ML 4.6, DV −5 and −7. Cells viability was assessed bytrypan blue (Sigma-Aldrich) after each transplantation session.

Study Design

The in-vivo experiment was performed on the 6-OHDA induced striatallesion model. Cells, or PBS as control, were transplanted on the sameday of the 6-OHDA injections, 50 minutes later, posterior to the lesionwithin the treated striatum. The experiment consisted of the followinggroups: the control group was treated with 6-OHDA and with PBS insteadof cellular treatment (n=10); MSC treated groups were treated witheither a high dose (450,000 cells, n=10) or a low dose (150,000 cells,n=11) of serum free medium treated MSC; NTF-SC group was treated witheither high dose (450,000 cells, n=10) or low dose (150,000 cells, n=11)of induced NTF-SC. Another group of untreated animals (n=7) were used ascontrols for the open field test.

For cell tracking purposes three different time points were analyzed byusing histology based study or in-vivo MRI. At the first time point, 7days post cellular treatment, 4 animals that were treated with high doseNTF-SC were sacrificed for histological evaluation only. For the secondtime point, on the 35^(th) day, we conducted an in-vivo MRI study onselected animals. These animals (n=3 from the control group and n=3 fromthe low dose NTF-SC treated group) were treated with cells that werepre-labeled with super-paramagnetic iron oxide particles (SPIOs, 5μg/ml, Feridex, Advanced magnetic, Cambridge, Mass., USA). SPIOs wereincubated with poly-L-lysine (1 μg/ml medium, 70-150 KD, Sigma-Aldrich)for one hour before adding to the medium on the last day of stage 1medium treatment. Cultures were washed with stage 2 medium of inductionafter 24 hours. The control group was treated with 1 μg of SPIOs in 6 μlof PBS (the same volume of the cell suspension). The last time point forcell tracking was at the end of the experiment, at 50 days aftertreatment day.

Immunosuppression was induced by daily subcutaneous administration of 15mg/Kg cyclosporine A (Novartis, Basel, Switzerland), starting one dayprior to cellular treatment and continued throughout the experiment.Animals received prophylactic antibiotic treatment with Enrofloxacin (50mg/Kg, Bayer, Germany) for five days from the first day of theexperiment.

Behavioral Tests

D-Amphetamine-induced rotational behavior was measured for 90 minutesfollowing i.p. administration of 2.5 mg/Kg (Sigma-Aldrich) using anautomated Rotameter device (San Diego Instruments, San-Diego, Calif.,USA). The net ipsilateral rotations were measured at 14, 28 and 42 dayspost cell transplantation.

Open field test was conducted at 7 days post treatment by introducingthe animals into a 50 cm² arena and videotaping the spontaneous behaviorof the rats for 30 minutes. The images were analyzed by EthoVision 3software (Noldus, The Netherlands).

MRI

Anesthesia was induced with 4% isoflurane in 95% O₂, and maintained with˜1-2% isoflurane (Vetmarket ltd., Petah-Tikva, Israel) at a flow rate of˜1 liter/minute. Respiratory rate was monitored throughout all theexperiments. Body temperature was maintained by circulating water at 37°C. MRI scans were performed on a 7.0 T/30 cm Bruker Biospec equippedwith a gradient system capable of producing gradient pulses of up to 400mT/m (Bruker Biospin, Karlsruhe, Germany). A body coil was used as thetransmit coil, and a rat quadurature coil was used as the receivingcoil. MRI experiments were performed on the 35th day posttransplantation and 6-OHDA injection. Scans included: T₂ weighted images(WI) RARE8 (TR/TE=3500/75 ms). The field of view (FOV) was 2.56×2.56,the matrix size was 256×128 zero filled to 256×256, and a slicethickness of 700 μm was chosen, 15 slices were collected. Additionally,three dimensional (3D) gradient echo (GE) images were collected (FLASH,TR/TE=150/14 ms, flip angle=15°) with a FOV of 2.56×2.56×0.48 and amatrix size of 128×96×24 (zero filleed to 128×128×32), resulting in aspatial resolution of 200×200×150 (μm)³. The images are presented as rawdata without any image processing.

Immunohistochemistry

At the end of the experiment (7 or 50 days post treatment) animals weretranscardially perfused with ice cold PBS following a solution of 4%paraformaldehyde and 4% sucrose in phosphate buffer (PB) according to aknown protocol (NeuroScience Labs, NSA, Knoxville, Tenn.). Brains wereimmersed in the perfusion solution for 24 hours in 4° C. followingcryoprotection in 30% sucrose for additional 48 hours before freezing.12 samples were processed by NSA (4 of the PBS group, 4 of the high MSCgroup, 4 of the high NTF-SC group and 4 animals treated with high doseof NTF-SC cells that were sacrificed after a week). These samples wereserially sectioned into 40 μm coronal sections and every 8^(th) sectionthroughout the striatum was dyed for human nuclei antigen and theadjacent section for tyrosine hydroxylase (TH).

The brains of animals treated with SPIOs labeled cells were sectionedaxially (8 μm) and dyed with Prussian blue stain (Sigma-Aldrich,according to manufacturer instructions) for the detection of Feparticles. Adjacent sections were immunostained with anti human nuclearantibody. Briefly, sections were microwave-boiled in a citrate bufferfor antigen unmasking, and then immersed in a blocking and permeabilitysolution (10% fetal calf serum, 2% bovine serum albumin, 1% glycine and0.05% Triton). Post blocking, the samples were incubated overnight withanti human nuclear antibody (1:200) in 4° C. Sections were dyed withanti mouse IgG conjugated to Alexa Fluor 568 (Invitrogen, 1:500). Nucleiwere counterstained with DAPI (1:500, Sigma-Aldrich). CD68 staining(1:500, Serotec, Oxford, UK) was conducted in a similar manner exceptthe antigen unmasking process and the use of biotinilated anti mouse IgG(ready to use, Zymed-Invitrogen) followed by streptavidin Alexa Fluor568 (1:500, Invitrogen).

Stereological Study

All sections stained for TH (n=4 animals from each group) werequantified for the area with a positive stain in the striatum. By usingan Olympus DP71 camera (Japan) at a X40 magnification, 2-3 images werephotographed to cover all or almost all the striatum of each side andeach animal. The images were then quantified by the ImagePro 5.1software that measured the total area of positive staining, according toa unified cutoff. The operator of the software was blind to the originof the images. The damage was calculated as the percent of theTH-positive area in the lesioned striatum divided by the TH-positivearea of the untreated contralateral side.

Dopamine Measurements by HPLC

Animals (n=5 each of the following groups: PBS, high dose MSC and highdose NTF-SC) were sacrificed by CO₂ and their brains were quicklyremoved and placed on ice. The striatae were dissected out and weighted.Each sample was sonicated in ice cold 1 ml of 0.1 M perchloric aciduntil homogeneity was achieved. The samples were centrifuged for 15minutes (12,000 rpm, 4° C.), and the supernatants were collected andtransferred onto a 0.2 μm nylon filter tubes (Corning). The samples werecentrifuged again (6,000 rpm, 5 minutes, 4° C.) and the filtrates werestored in −80° C. until analyzed. An aliquot of the filtrate wasinjected into the HPLC system (Waters, Milford, Mass., USA) equippedwith a C18 reverse phase, 3μ LUNA column (100 mm×2 mm, Phenomenex,Torrance, Calif., USA). The sample was eluted by a mobile phase made of25 mM NaH₂PO₄, 50 mM Na-citrate, 0.03 mM EDTA, 10 mM diethylamine HCland 2.2 mm sodium octyl sulphate (pH 3.2), 30 ml/L methanol and 22 ml/Ldimethylacetamide at a flow rate of 0.4 ml/min. The dopamine peak wasdetermined by electrochemical detection at a potential of 0.6 V. Thedopamine content in the sample was calculated by extrapolating the peakarea from a standard curve (range 1-200 pg of dopamine) constructedunder the same conditions during each run by the Maxima Workstation(Waters). The results were normalized to the sample weight.

Statistical Analysis

The results are expressed as means±standard error. Student's t-test wasused to compare means of two groups. Comparisons between several groupswere done by ANOVA with Scheffe's post-hoc analysis. Repeated tests(amphetamine-induced rotations) were also analyzed by repeated ANOVAtest. Statistical calculations were performed using SPSS v. 13.

Results

Transplanted Human NTF-SC Attenuate 6-OHDA Induced Rotational Behaviorin Rats

Overall, the treated animals of all groups tolerated well the varioustherapies including immunosuppression. Of 56 animals, 2 died within theexperimental follow up, one of peritonitis, and the other of an unknowncause. The animals failed to gain weight for 14 days post treatment,regardless of the group tested (excluding healthy controls for the openfield test), and from that point on, almost all animals gained weight ina similar manner.

The control (PBS) group demonstrated a statistically significantincrease of amphetamine-induced ipsilateral rotations on the 14^(th) daypost lesion (2.71±0.79 net ipsilateral rotations per minute) and the28^(th) day (4.11±0.86), reaching a plateau on the 42^(nd) daymeasurement (4.74±1.07) suggesting that the pathological process ofdeath of nigrostriatal dopaminergic nerve terminals was progressive innature for at least 28 days.

There were no statistically significant differences between thedifferent cell doses, regardless of the type of treatment and the day ofmeasurement. In the untreated MSC low dose group it was found that theanimals rotated at a rate of 3.54±0.63, 2.61±0.55 and 3.25±0.75 netipsilateral turns per minute on days 14, 28 and 42 post treatmentrespectively, while the MSC high dose treated group showed lower but notstatistically significant measures: 2.05±0.41, 2.22±0.52 and 2.30±0.78at the same time intervals. The NTF-SC treated group had shown the sametrait between the doses: 1.19±0.6, 1.29±0.44 and 2.07±0.81 in the lowdose treated group compared to 2.12±0.42, 1.49±0.32 and 1.67±0.52 in thehigh dose treated group. Hence, the high and low dose groups werecombined for another statistical analysis. It was found that bothcellular treatment types produced a non-progressing effect on therotational behavior. The NTF-SC group demonstrated a lower result in thefirst measurement fourteen days post-treatment, although it notstatistically significant different from the other groups. It had astatistically significant better result than the control group on thetwo later measurements (2.45±0.54 and 2.86±0.54 for the combined MSCtreated group on days 28 and 42 post treatment, respectively, comparedto 1.46±0.37 and 2.16±0.37 for the combined NTF-SC treated group at thesame time intervals, p<0.05 compared to the PBS group). In summary, theMSC treated group did not demonstrate a statistically significantimprovement as compared to the control (PBS) group. In contrast, for thecells undergoing the novel induction based treatment, the NTF-SC groups,a marked decrease of 25% and 45% after 14 and 28 days posttransplantation was noted (FIG. 5A).

All 6-OHDA treated animals demonstrated overall motor hypoactivity inthe open field test on the 7^(th) day post treatment. The control groupactivity indices were similar to those of the MSC treated group. Thetotal distance walked by the combined low and high dose NTF-SC treatedanimals was 3667 cm on average, which was 22% higher compared to thecontrol group (2989 cm) and 17% higher than the combined low and highdose MSC treated group (3119 cm) on average. However, there was nostatistically significant difference between the groups (p=0.054) (FIG.5B).

Transplantation of Human NTF-SC Resulted in a Greater Preservation ofTH-Positive Area in the 6-OHDA Lesioned Striatum

The methodology of the stereological study is illustrated in FIGS.6A-6E. It was found that 6-OHDA-induced lesions with this specificprotocol decreased the striatal TH-positive area by more than 5-fold incomparison to the control hemisphere (15.26±2.95% of the contralateralstriatum). Both types of cellular treatment, MSC and NTF-SC,demonstrated a protective effect. However, only the NTF-SC yielded astatistically significant higher TH-positive area as a percent of theuntreated side (31.15±6.27%), by increasing it more than twice incomparison to the control group, while MSC treatment failed to induce astatistically significant difference (23.11±3.5%, FIGS. 7A-7D).

Interestingly, when subdividing the different areas of the striatum intosections of approximately 1 mm thick, it was found that the NTF-SC werebeneficial in the anterior, rather than the posterior area of thestriatum, the region of transplantation (FIG. 7E).

Cellular Transplantation Inhibited 6-OHDA-Induced Dopamine Depletion inthe Lesioned Striatae

It was found that both types of cellular treatments, i.e. MSC andNTF-SC, prevented the falls of striatal dopamine levels induced byneurotoxin injection by more than 2.5-fold compared with the controlgroup (HPLC measurement of the levels of dopamine in the whole lesionedstriatum, was defined as a percentage of the untreated side). While thecontrol group (n=5) lesioned striatae contained only 21.3±3.8% dopaminemeasured in comparison to the untreated side, MSC treated animals (n=5)had on average 68.7±8.6% and the NTF-SC treated group (n=5) had72.4±16.4% striatal dopamine levels compared to the intact contralateralstriatum. Only measurements from the NTF-SC treated group reached astatistically significant difference compared to the control group(p<0.05).

Tracking of Transplanted Cells by Histology and in-Vivo MRI

Cell tracking was done at three different time points, by applyingin-vivo MRI and three different histological assessments. In-vivo MRIwas conducted on selected animals treated with iron particles-labeledcells (or SPIOs only as controls, n=3 each) on day 35 post treatment.The 3D T₂* weighted images demonstrated two distinct hypointense regionsfor the PBS treated group: the PBS/SPIO injection site and the 6-OHDAinjection sites (FIG. 8A). The latter probably resulted from bleeding inthe site of 6-OHDA injection. No other hypointensities could be seen inthe area between the two injections. In contrast, the 3D T₂* weightedimages of the cell-treated group demonstrated a migration pathway fromthe site of NTF-SC injection to the striatum (FIG. 8B-8C). The 2D T₂weighted image is shown in FIG. 8G for anatomical reference.Histological staining with Prussian blue for the iron particles was inexcellent agreement with hypointensities in the MR images (FIG. 8D).

Further histological studies aimed at the search for human nuclearantigen. Four randomly selected animals treated with the higher dose ofhuman NTF-SC were sacrificed for histological studies at the first timepoint, i.e., 7 days after they were treated with 6-OHDA. The brains ofthe animals were serially sectioned as described. Using the NSA methods,a large cluster of cells around the injection site were observed in eachof these animals. In three out of the four specimens, cells were foundto be present along 620-1920 μm from the site of transplantation inadjacent sections, indicating the beginning of cellular migration. Inorder to quantify the survival rate of the cells, all the cells withnormal morphology were manually counted, (i.e. those that weresmooth-looking and not comprising egmented nuclei with a positive dye.This measurement revealed that only 0.34±0.1% of the cells survived oneweek post transplantation.

On the last day of the experiment, i.e. 50 days post-treatment, NSAmethods were used on 4 randomly selected animals from the PBS controlgroup, the high MSC-treated group and the NTF-SC-treated group. Thisexamination revealed almost no cells. In fact, around thetransplantation site, only remnants of cells and signs of old bleedingwere found. After sectioning other brains into 8 μm sections and using amore sensitive fluorescent based stain, a small minority of thetransplanted human cells after 50 days were observed. The staining wasconducted on axial sections adjacent to those in which a positivePrussian blue dye was found, and all cells positive for human nuclearantigen were demonstrated along the migration path. However, using thefluorescence method, the present inventors could not find cells alongthe entire migration pathway indicated by the Prussian blue stain,indicating that iron deposits did not necessarily indicate living cellsby the last day of the experiment (FIGS. 8E-8G). CD68 staining for theidentification of macrophages in the route of the cells was negative(data not shown). In summary, migration of human cells within thelesioned rat striatum was noted by in-vivo MRI and by histologicalanalysis; a small minority of the cells was found to survive for atleast 50 days.

CONCLUSIONS

In this report, a robust induction protocol of adult human bone marrowderived MSC into NTF-SC is described. Such cells produce and releaseseveral NTFs including BDNF and GDNF. These cells conditioned mediarescued 6-OHDA-treated neuroblastoma cells. In cyclosporinized rats withunilateral striatal 6-OHDA lesions, ipsilateral transplantation of humanNTF-SC was beneficial and partially attenuated amphetamine-inducedrotations and other abnormal behavior, striatal dopamine levelsreduction, as well as the loss of TH-immunoreactive nerve terminalnetwork. It was also found that the NTF-SC migrated along the corpuscallosum around the striatal 6-OHDA lesion into the anterior striatum,instead of migrating directly to the lesion area.

It was found that the NTF-SC secrete significantly higher levels of NTFscompared to untreated MSCs. This protocol increased the levels of BDNFand GDNF 2.3- and 5.8-fold, respectively, as compared to control humanMSC. Immunocytochemical studies revealed that the induction process isrobust, since almost all cells are positive for the tested NTF.

Cell-based delivery of NTFs may be potentially superior to directinfusion of NTFs since it does not require instruments oftransplantation such as permanent catheters, rather than a singlesurgical procedure. The presently proposed medium-based induction mayalso be superior to viral vector delivery for achieving geneticover-expression since it circumvents safety problems. Moreover, thepresent inventors used xeno-free media for both the production of theMSC culture and for their induction procedure until transplantation,rendering the present method more practical and acceptable for clinicaluse. The platelet-based growth medium did not alter the basicmesenchymal characteristics of the MSC in terms of CD markers andmesenchymal lineage differentiation.

Human neuroblastoma cells exposed to 6-OHDA are used as an in-vitromodel for PD, due to similar cellular processes that occur in thedegenerating dopaminergic neurons, such as oxidative stress andapoptosis. The conditioned media of MSC and NTF-SC protected theneuroblastoma cell line from a 6-OHDA induced cell death. Although bothMSC and NTF-SC demonstrated beneficial effects, NTF-SC-based treatmentwas more potent in protection from the 6-OHDA, specifically in higherinsult concentrations. The conditioned media used in this experimentconsisted of a serum-free media placed on the cells 24 hourspost-induction. Therefore, it consisted of only the factors secreted bythe cells, and not induction media. This strongly implies that themechanism underlying the observed protection is the presence of secretedNTFs.

In order to perform in-vivo testing for the assumption that NTF-SC-basedtreatment is beneficial in a PD animal model, the present inventorsfirst calibrated the well-established 6-OHDA-induced hemiparkinsonianrat model by injecting the specific dose of 6-OHDA into two striatallocations. As opposed to injections into the medial forebrain bundle,the present inventors used a relatively mild and progressive model ofPD, probably representing an early phase of PD in human subjects. Thecellular transplantation treatment was given on the day of 6-OHDAinjection, therefore aiming at neuroprotection, rather than atregeneration of already-lost dopaminergic terminals. The animals wereexamined by two well documented behavioral tests: amphetamine-inducedrotations and spontaneous motor activity in an open field.

The effect of NTF-SC treatment in terms of amphetamine-induced rotationswas comparable to that found by some researches who utilized dopamineproducing cells in the same animal model. The present finding thatNTF-SC transplantation reduces the amphetamine-induced rotations by 40%as compared to the control group indicates that the present therapeuticstrategy, of employing stem cells as inducers of neuroprotection, isefficient and comparable to dopaminergic cell replacement strategies.The NTF-SC treated group also demonstrated a positive trend in the openfield test, whereas the MSC did not alter the hypoactive behaviorcompared to the PBS treated group.

Following the 6-OHDA lesion, the striatal TH immunoreactive fiber areaof the MSC treated group was larger than in the PBS-treated animals.However, only the NTF-SC treated group demonstrated a statisticallysignificant lesser destruction of DA nerve terminals attested by amarkedly larger TH-positive area compared to the PBS treated group. Asstated, only the most anterior part of the striatum benefited from theNTF-SC treatment, at a distance from the transplantation site. It isinteresting to note that in that area of the striatum, the damage wasnot as severe as in the posterior section. Hence, NTF-SC may prove morebeneficial in a moderately injured tissue, but not in a state ofcomplete or near-complete loss of dopaminergic nerve terminals. Themigratory path of the transplanted cells was observed through the corpuscallosum to the anterior parts of the striatum and not directly from thetransplantation site into the lesion. This might be an indication thatthe surviving cells affected the anterior, rather than the posteriorstriatum.

Although MSC partially rescued dopamine levels, only the NTF-SC-treatedgroup demonstrated a statistically significant lesser decreases ofstriatal dopamine levels which makes this treatment superior to theconventional non-induced MSC-based therapy.

An important issue in stem cell research is the survival of thetransplanted cells in-vivo. In this work the present inventors employeddifferent methods over several time points in order to address thissubject. Using in-vivo MRI the present inventors were able todemonstrate the migration capacity of otheur cells, which was highlycorrelated to Fe histological staining. A migratory route was observedthat bypassed the lesion site along the corpus callosum and led into theanterior striatum. Such cellular migration proves two major points:firstly, that the migrated cells survived in the CNS; and secondly, thatthe cells moved towards a specific signal traveling along a specificroute.

In an attempt to quantify the number of surviving cells, a stereologicalmethod at 7 and 50 days post treatment was used. When applying thisrelatively insensitive but reproducible method, it was found that thevast majority of the transplanted cells were rejected within a week posttransplantation, and that no cells were found in the treated striatumafter 50 days, even though immunosuppressive therapy was given. This lowsurviving rate is probably due to immune rejection, and to some extentimmediate cellular death due to shear forces when passing though a thinsyringe into the tissue. When a more sensitive method was used, with ahigher signal to noise ratio, the surviving cells were highlighted, asthe tissue was sliced into thin 8 μm sections and a fluorescent dye wasused. Moreover, the survival of the cells in the specific location inwhich they were found was supported by its high correlation to the MRimages and the Prussian blue stain for iron particles in the specificspecimens in which we used SPIOs labeled cells. The absence ofmacrophages (CD68 expressing cells) along the migration route isadditional evidence of the survival of the migrating transplanted cells.It is therefore implied that although only a minority of the cellssurvived throughout the experiment, they were sufficient to induce thedescribed beneficial effect in the lesioned striatum. Another strongpossibility is that the transplanted cells exerted their full beneficialefficiency soon after their placement into striatum and that there wasno further need for their entire presence later on.

Example 3 Human Mesenchymal Stem Cells Differentiated into NeurotrophicFactors Secreting Cells Perform Glutamate Uptake

The present study was performed in order to ascertain whether inductionof adult hMSC into NTF-SC increases their ability to perform glutamateuptake and further to ascertain whether this uptake was significantlyreduced by glutamate uptake inhibitors.

Materials and Methods

In-vitro differentiation: Donor MSCs were grown and differentiated asdetailed in Example 1 herein above.

Glutamate uptake: Glutamate uptake was assessed using [³H] D-aspartate(Amersham Pharmacia Biotech, Roosendaal, Netherlands), a transportableanalogue of L-glutamate, which does not interact with glutamatereceptors and is not metabolized. Differentiated and non-differentiatedMSC were plated on poly-L-lysine coated 24 well plates, at aconcentration of 2.5×10⁴ cells per well. Plated cells were maintained inserum free medium (DMEM supplemented with SPN and L-Glutamine asspecified for platelets medium above.) for 24 hours. Cells were rinsedtwice with 0.5 ml Krebs buffer (25 mM HEPES pH 7.4, 4.8 mM KCl, 1.2 mMKH₂PO₄, 1.3 mM CaCl₂, 1.2 mM MgSO4, 6 mM glucose and 140 mM NaCl)preheated to 37° C. Cells were incubated in 0.5 ml of preheated Krebsbuffer and [³H] D-aspartate at a final concentration of 50 nM. Uptakewas stopped after 20 minutes by three rinses with cold Na⁺ free Krebsbuffer (25 mM HEPES pH 7.4, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 1.2mM MgSO₄, 6 mM glucose and 120 mM choline chloride- NaCl was replacedwith choline chloride at the same osmolarity). Cells were lysed with 0.5ml of 1 M NaOH. The radioactivity of 350 μL was determined by liquidscintillation counting. 150 μL of lysate were removed for proteinconcentration tests performed using Bradford assay (Bio-Rad LaboratoriesLtd.). Na+ free tests were performed in 0.5 ml Na+ free Krebs bufferpreheated to 37° C. Glutamate uptake inhibition was performed using0.314 μM L-trans-Pyrrolidine-2,4-dicarboxylic acid (t-PDC, Sigma-AldrichSt. Louis), it was add after the second rinse and was incubated for 15minutes prior to [³H] D-aspartate addition. Competitive inhibition wasachieved using cold (non-radioactively labeled) D-methyl-aspartate at afinal concentration of 50 nM on top of the labeled [³H] D-aspartate.

Results

Functional glutamate transport in cultured MSC and NTF-SC was evaluatedby measuring the amount of [³H] D-aspartate taken up by the cells. [³H]D-aspartate is a transportable glutamate analogue that does not interactwith glutamate receptors and is not metabolized. Thus, this assayprovides a strong indication of the cells ability to perform glutamateuptake.

NTF-SC perform considerable [³H] D-aspartate uptake as shown in FIG. 9.[³H] D-aspartate uptake was significantly increased when MSC wereinduced into NTF-SC (P<0.0001). When the assay was performed in a Na⁺free buffer, uptake was reduced by 57.5%. Furthermore, competitiveinhibition by D-methyl-aspartate reduced uptake by 86.76%, whileinhibition by t-PDC a non-specific glutamate uptake inhibitor reducedglutamate uptake by over 90%.

These results suggest that induction of MSC into NTF-SC increasesglutamate uptake, that NTF-SC perform significant glutamate uptake andthat this uptake involves both Na⁺ dependent and independent transport.

CONCLUSIONS

This study indicates the ability of bone marrow derived stem cells totake up glutamate. This ability is drastically improved after inductionof hMSC into NTF-SC. Glutamate is extremely ubiquitous in the human CNS,however overexposure to glutamate is highly toxic. Glutamateneurotoxicity has long been known to contribute to the pathogenesis ofneurological disorders such as cerebral ischemia, AD, PD, HD, epilepsyand ALS.

Transplantation NTF-SC cells near the insult sight could allow neuronalprotection from glutamate toxicity by regulating extracellular glutamatelevels without interfering with proper glutamate neuronal transmission.Glutamate release inhibitors and receptor antagonists are already beingused to treat several neurological conditions. Riluzole, a drug used inthe treatment of ALS and obsessive compulsive disorder (OCD) reducesglutamate release. Glutamate antagonists are being tested in stroke inthe hope of limiting the size and severity of the ischemic insult andare already employed in several antiepileptic drugs. Specific Glutamatereceptor antagonists are currently undergoing clinical tests in thetreatment of AD. Nevertheless, this solution interferes with the naturalfunctions of glutamate, increasing the plausibility of severe sideeffects, reduced by the use of NTF-SC.

The combined therapeutic effect of neurotrophic factors and glutamateuptake provides the best survival chances of degeneration prone neuronsin each and every one of the different neurological disorders.

Example 4 Further Characterization of Neurotrophic-Factor-SecretingCells (NTF-SCs)

Materials and Methods

Real-time reverse transcription polymerase chain reaction: Real-time PCRof GFAP was performed in an ABI Prism 7700 sequence detection system(Applied biosystems) using Sybr green PCR master mix (Appliedbiosystems). GAPDH gene served as a valid reference ‘housekeeping’ genefor transcription profiling.

The PCR was performed in a total volume of 20 μl containing 1 μl ofcDNA, 1 μl each of the 3′ and 5′ primers (final concentration of 500nmol/L each), 10 μl of Absolute™ QPCR SYBR® Green ROX Mix and 8 μl ofDEPC water.

The amplification protocol was 40 cycles of 95° C. for 15 sec followedby 60° C. for 1 min each.

Immunocytochemistry: For immunochemistry analysis, cells were grown on12 mm round poly-L-lysine coated glass coverslips. At the end of theexperiment the medium was removed, cells were fixed withparaformaldehyde 4% (v/v) for 20 minutes at room temperature andpermeabilized thereafter with 0.25% Triton X-100 (v/v) in 0.1 M PBS for20 minutes. Non-specific binding was blocked by incubating the cells ina 0.1 M PBS solution containing 5% normal goat serum (NGS) and 1% bovineserum albumin (BSA) (Sigma) for 1 hour at 37° C. Subsequently, the cellswere incubated in a 0.1 M PBS solution containing 0.25% Triton X-100(v/v), 5% NGS and 1% BSA with primary antibodies i.e. rabbit anti-glialfibrillary acidic protein (GFAP) 1:100 (DAKO), mouse anti human nuclearh-Nuc 1:30 and mouse anti S100β 1:200 (Sigma-Aldrich, St. Louis, Mo.,USA). Secondary antibodies were added for 1 hour and subsequentlystreptavidine-Alexa-488 conjugated goat anti-rabbit IgG antibody 1:200(Molecular Probes, Eugene, Oreg., USA). For other staining, Alexa-488conjugated goat anti-rabbit IgG antibody 1:200 andRodamine-Rx-conjugated 1:200 (Jackson ImmunoResearch Laboratories, WestGrove, Pa., USA) were diluted in 0.1 M PBS solution containing 0.25%Triton X-100 (v/v), 5% NGS and 1% BSA and were applied for 1 hour atroom temperature. Nuclei were stained for 5 minutes with the nuclear dyeDAPI 1:200 (Sigma, Aldrich). Following three rinses in PBS, thepreparations were mounted in Antifaiding (Sigma, Israel) and examinedusing a fluorescent microscope coupled to a CCD camera (T.I.L.L.photonics, Martinsried, Germany). Excitation wavelengths (488, 405 and568 nm for Alexa 488, DAPI and Alexa 568, respectively) were generatedusing a Xenon lamp coupled to a monochromator (T.I.L.L. photonics,Martinsried, Germany). Digital images were acquired using appropriatefilters and combined using the TILLvisION software.

Results

The results are illustrated in FIGS. 10A-10C,

Specifically, FIGS. 10A-10C show the increase in GFAP in thedifferentiated cells of the present invention, wherein more than 90% ofthe cells express GFAP (FIG. 10C). FIGS. 11A-11C show that more than 90%of the differentiated cells of the present invention express S100.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A pharmaceutical composition comprising a culturemedium as the pharmaceutically acceptable carrier and a population ofnon-genetically modified human cells as the active agent, wherein saidpopulation of non-genetically modified human cells have beendifferentiated ex vivo from mesenchymal stem cells as the active agent,wherein said population of non-genetically modified human cells expressat least one mesenchymal stem cell marker and secrete both brain-derivedneurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF),wherein said population of non-genetically modified human cells do notsecrete nerve growth factor (NGF), wherein said at least one mesenchymalstem cell marker is selected from the group consisting of CD73, CD90 andCD105.
 2. The pharmaceutical composition of claim 1, wherein saidpopulation of non-genetically modified human cells, further express atleast one additional neurotrophic factor.
 3. The pharmaceuticalcomposition of claim 2, wherein said at least one additionalneurotrophic factor is selected from the group consisting ofneurotrophin-3 (NT-3), neurotrophin-4/5, Neurturin (NTN), Persephin,artemin (ART), ciliary neurotrophic factor (CNTF), insulin growthfactor-I (IGF-1) and Neublastin.
 4. The pharmaceutical composition ofclaim 2, wherein said culture medium comprises DMEM.
 5. A method oftreating a CNS disease or disorder comprising administering to anindividual in need thereof a therapeutically effective amount of thepharmaceutical composition of claim 1, thereby treating the CNS diseaseor disorder.
 6. The method of claim 5, wherein the CNS disease ordisorder is a neurodegenerative disease or disorder.
 7. The method ofclaim 5, wherein the CNS disease or disorder is selected from the groupconsisting of a motion disorder, a dissociative disorder, a mooddisorder, an affective disorder, an addictive disorder and a convulsivedisorder.
 8. The method of claim 5, wherein the neurodegenerativedisorder is selected from the group consisting of Parkinson's, multiplesclerosis, epilepsy, amyotrophic lateral sclerosis (ALS), stroke,autoimmune encephalomyelitis, diabetic neuropathy, glaucomatousneuropathy, Alzheimer's disease and Huntingdon's disease.
 9. The methodof claim 5, wherein said non-genetically modified human cells areautologous to said subject.
 10. The method of claim 5, wherein saidnon-genetically modified human cells are non-autologous to said subject.11. The method of claim 5, wherein said non-genetically modified humancells are allogeneic to said subject.
 12. The method of claim 5, whereinsaid administering to the subject comprises administering to the centralnervous system (CNS) of the subject.
 13. The method of claim 5, whereinsaid administering to the subject comprises administering to the musclesof the subject.